Food Chemistry 167 (2015) 463–467
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Relative quantitation analysis of the substrate specificity of glutamyl endopeptidase with bovine a-caseins Yi-shen Zhu a,b,⇑, Phanindra Kalyankar b, Richard J. FitzGerald b a b
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China Department of Life Sciences, University of Limerick, Limerick, Ireland
a r t i c l e
i n f o
Article history: Received 19 February 2014 Received in revised form 6 June 2014 Accepted 6 July 2014 Available online 11 July 2014 Keywords: Glutamyl endopeptidase Bovine a-caseins’ digest Substrate specificity Isobaric tag for relative and absolute quantification LC–MS/MS
a b s t r a c t A bovine a-caseins’ preparation digested with glutamyl endopeptidase (GE) at 37 and 50 °C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Incubation temperature was shown to affect protein digestion. MS analysis of the digestion products indicated that phosphorylated peptides were less sensitive than non-phosphorylated peptides according to the MS intensities. GE hydrolysed Glu(51)-Tyr(52) and Glu(50)-Glu(51) in Glu(49)-Glu(50)-Glu(51)-Tyr(52) of bovine as1-casein. The results herein helped to confirm the precise process of a-caseins’ hydrolysis with GE, which is significant for quantifying the release of bio- and techno-functional peptides. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hydrolysed milk proteins are widely applied in food industry for many purposes, e.g., as ingredients in infant formula and in parenteral nutrition (Meisel, 2004, 2007; Nongonierma & FitzGerald, 2012; Power, Jakeman, & FitzGerald, 2013). a-Caseins (as1- and as2-casein) are major proteins in bovine milk accounting for approximately 38% of overall milk protein. Analysis of the digestion of a-caseins is therefore important for nutritional studies of dairy protein. Glutamyl endopeptidase (GE) is a chymotrypsin-like serine protease, mainly found in Bacillus species, that specifically cleaves peptide bonds between negatively charged amino acid residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. has reported that GE is more efficient in the hydrolysis of caseins compared to whey proteins (Madsen & Qvist, 1997). The substrate specificity of GE with bovine a-caseins’ preparation has been qualitatively studied (Kalyankar, Zhu, O’Keeffe, O’Cuinn, & FitzGerald, 2013). However, to our knowledge, the hydrolysis process was not quantitatively characterised. A quantitative analysis is necessary to provide more detailed information on the GE a-caseins’ hydrolysis process. Isobaric tag for relative and absolute quantification (iTRAQ) is one of the two pioneering silent isotope incorporation methodologies (Wright, Gan, Chong, & Pham, 2007). Both the identification of ⇑ Corresponding author. Tel.: +86 18900660563; fax: +86 25 58139910. E-mail address: [email protected] (Y.-s. Zhu). http://dx.doi.org/10.1016/j.foodchem.2014.07.017 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
proteins/peptides and their quantification can be achieved in a single injection (Sickmann, Burkhart, Vaudel, Zahedi, & Martens, 2011). In food science, the iTRAQ labelling was applied to quantitative proteomic analysis of bacterial enzymes in ripening cheese (Jardin, Molle, Piot, Lortal, & Gagnaire, 2012). However, quantitative analysis of enzymatic digestion of milk proteins using iTRAQ methodologies does not yet appear to have been reported in the literature. This work reports the quantitative investigation of the substrate specificity of GE with bovine a-caseins, the main proteins in bovine milk. GE was purified from Alcalase™ and its specificity was investigated using a-caseins. The hydrolysates of a-caseins digested with GE at different times at 37 and 50 °C were quantitatively analysed using iTRAQ technology. 2. Materials and methods 2.1. Materials The iTRAQÒ Reagents – 4plex Applications Kit and ICATÒ Cation Exchange Buffer Pack were supplied from Applied Biosystems (Toronto, Canada). All the other analytical reagents were obtained from Sigma Aldrich (Dublin, Ireland). 2.2. Protein purification and digestion GE was purified from Alcalase™ 2.4 L with hydrophobic interaction chromatography (HIC) of phenyl SepharoseÒ and
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Y.-s. Zhu et al. / Food Chemistry 167 (2015) 463–467
ion-exchange (IEX) of HiTrap™-CM FF (Kalyankar, Zhu, O’Keeffe, O’Cuinn, & FitzGerald, 2013). The a-caseins were purified from bovine acid caseinate (Arrabawn Co-op Society Ltd, Tipperary, Ireland) following the procedure reported by Kalyankar (2011). The aqueous solution (2 mL, 1.5% (w/v)) of GE (256 nmol min 1 mL 1) digested a-caseins were incubated at 37 and 50 °C. Samples (250 lL) were withdrawn at 0, 15, 60, 120 min and were diluted with 450 lL of 0.1% (v/v) formic acid in HPLC grade H2O (Kalyankar et al., 2013). 2.3. iTRAQ labelling Each of diluted samples was labelled with iTRAQ reporter ions according to the manufacturer’s protocol (Applied Biosystems, Toronto, Canada). The brief procedure was described as: 15 lL of ethanol diluted iTRAQ reagent and 30 lL dissolution buffer were transferred to each of the 5 lL diluted samples (sampling at 0, 15, 60 and 120 min from the GE digestion); the mixture was incubated at room temperature (20 °C) for 1 h; samples were incubated for 1 h at room temperature; ethanol was removed by vacuum vortex for 1 h. The four labelled samples were mixed in a 1:1:1:1 (v/v) ratio, resulting in 27 lg of iTRAQ-labelled digest. The samples were loaded and washed on an ICATÒ cation exchange cartridge according to the manufacturer’s protocol (Applied Biosystems) to remove the excess iTRAQ reagent. 2.4. Liquid chromatography–mass spectrometry (LCMS) analysis of iTRAQ labeled GE digests of a-caseins Tandem mass spectrometry of iTRAQ-labelled samples was performed on a micrOTOFQ-II system (Bruker Daltonics, Bremen, Germany) coupled to an Ultimate 3000 nano-flow high performance liquid chromatography (HPLC) (Dionex, Sunnyvale, USA). All iTRAQ samples were first desalted online using a C18 PepMap 100 precolumn cartridge (Dionex) using 0.1% TFA at flow rate of 25 lL/min for 30 min. Desalted samples were eluted to a 15 cm, 75 mm ID C18 PepMap analytical column (Dionex) in 0.1% formic acid. Elution was then performed using a predefined 60 min LC gradient program (2–40% acetonitrile, (ACN) containing 0.1% formic acid) and further eluted for 5 min at 95% ACN. Duplicate samples of the different a-caseins’ digests were analysed on separate
LCMS/MS runs. MS measurements were all performed on a predefined 50–2400 m/z acquisition window at 2500 TOF summations. 2.5. Data analysis Tandem MS data were processed via Compass Data Analysis v 4.0 SP4 (Bruker Daltonics). Parameters of peak finder were set to S/N at least 3 and minimum 10 counts intensity. Deconvolution of MS and MS/MS charges was set to between 200 and 2500 m/z, maximum of 5+ for MS and maximum of 3+ for MS/MS spectra. All iTRAQ data were searched against the NCBInr database (downloaded at Nov 11th, 2010). The iTRAQ modifications were set to include 4-plex iTRAQ mass shifts (iTRAQ ions were labelled at Lys (K), Tyr (Y) and N-term), phosphorylation of serine and threonine (+80 Da, Ser(S)/Thr(T)) and oxidation of methionine (+16 Da, Met(M)) as variable modifications. Mass tolerances for all identifications were set to 0.06 Da for MS and 0.1 Da for MS/MS. Peptide level filters were set to a MASCOT score of 20 and at a p 6 0.05 (Zhang, Ficarro, Li, & Marto, 2009). The identified iTRAQ peptides (MS/MS) were automatically calculated using WARP-LC 1.2 (Bruker Daltonics). To detect low molecular mass peptides manually, masses were searched against theoretical peptide sequences using DataAnalysis, BioTools and Sequence Editor Software packages (Bruker Daltonics). The peptide mass and fragment mass tolerances were both set to ±0.1 Da. 3. Results In the 4-plex iTRAQ labelled peptides, the fragments released from CID were the reporter ions at 114.1, 115.1, 116.1 and 117.1 m/z. The fragmentation spectrum of f15–30 of as1-casein is shown by way of example in Fig. 1. The sequence coverage was 56% and 60% for the identified as1-casein peptides at 37 and 50 °C, respectively (Tables 1 and 2). The sequence coverage was 26% and 30% for the identified as2-casein peptides at 37 and 50 °C, respectively (Tables 1 and 2). The false discovery rates (FDR) were less than 5%. The identified sequences of the iTRAQlabelled samples were combined to provide greater coverage and precise quantification (Wright, Chong, Gan, & Pham, 2006). The sequence coverage data shows that such a high temperature during digestion may increase the extent of hydrolysis, so that the
Fig. 1. The fragment ion spectra and the sequence with identified b and y ions of iTRAQ-labelled as1-casein f15–30 in the a-caseins’ hydrolysate sample digested by glutamyl endopeptidase at 37 °C with the quantification of the iTRAQ reporter ions. Note: V indicates that the N-terminal of valine was iTRAQ-labeled.
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Y.-s. Zhu et al. / Food Chemistry 167 (2015) 463–467 Table 1 iTRAQ-labelled peptide sequences identified upon incubation of a-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 50 °C. Fragment
Peptide sequences
Ion selected for MSMS
MASCOT score
115/114
116/114
117/114
f(15–30)
E-VLNENLLRFFVAPFPE-V
1025.0681 (2)
101.23
0.81
1.13
0.60
f(15–39)
E-VLNENLLRFFVAPFPEVFGKEKVNE-L
806.6994 (4)
75.11
0.82
0.53
0.31
as1-Casein
f(19–30)
E-NLLRFFVAPFPE-V
797.4491 (2)
60.03
1.03
1.57
2.14
f(19–35)
E-NLLRFFVAPFPEVFGKE-K
766.7678 (3)
31.37
1.16
1.79
1.95
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
923.5174 (3)
119.38
1.88
1.50
0.84
f(31–39)
E-VFGKEKVNE-L
741.4380 (2)
29.13
1.57
3.18
3.33
f(40–55)⁄
E-LSKDIGS⁄ES⁄TEDQAMoE-D
735.3034 (3)
41.37
0.80
1.01
1.51
f(56–63)⁄
E-DIKQM°EAE-S
634.3257 (2)
27.18
1.74
5.08
5.75
f(71–84)⁄
E-IVPNS⁄VEQKHIQKE-D
721.0629 (3)
56.69
1.07
10.69
11.77
f(71–89)⁄
E-IVPNS⁄VEQKHIQKEDVPSE-R
848.7698 (3)
37.95
2.41
11.04
4.26
f(71–89)⁄
E-IVPNS⁄VEQKHIQKEDVPSE-R
896.8038 (3)
47.07
2.10
5.10
4.77
f(90–96)
E-RYLGYLE-Q
529.2936 (2)
34.61
1.37
4.15
5.68
f(97–110)
E-QLLRLKKYKVPQLE-I
730.1350 (3)
28.59
0.88
2.36
2.67
f(119–125)
E-RLHSM°KE-G
402.2285 (3)
21.49
1.05
2.20
2.78
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE-L
737.8914 (4)
30.1
1.08
2.42
2.92
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE-L
773.9169 (4)
61.46
1.46
2.49
3.13
f(119–141)
E-RLHSMKEGIHAQQKEPM°IGVNQE-L
777.9156 (4)
44.57
2.45
3.39
2.55
f(119–141)
RLHSM°KEGIHAQQKEPM°IGVNQE-L
781.9143 (4)
23.68
1.18
3.57
2.92
f(126–141)
E-GIHAQQKEPMIGVNQE-L
689.7014 (3)
87.91
0.80
2.67
4.34
f(142–148)
E-LAYFYPE-L
1046.5315 (1)
35.32
1.46
2.20
2.84
f(13–23)
E-S⁄IISQETYKQE-K
847.4180 (2)
55.24
0.57
0.28
0.13
f(24–33)
E-KNMAINPS⁄KE-N
548.6232 (3)
21.54
3.11
3.96
5.93 3.08
as2-Casein
f(43–51)
E-VVRNANEEE-Y
602.3079 (2)
26.34
0.86
2.31
f(64–84)⁄
E-VATEEVKITVDDKHYQKALNE-I
716.3948 (4)
22.73
1.11
2.72
1.20
f(64–84)⁄
E-VATEEVKITVDDKHYQKALNE-I
752.4203 (4)
37.38
1.00
2.33
2.45
f(69–84)
E-VKITVDDKHYQKALNE-I
620.1107 (4)
43.95
1.50
4.63
5.80
f(134–145)
E-NSKKTVDM°ES⁄TE-V
584.9370 (3)
51.06
1.03
0.89
1.66
Note: S⁄ represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labelled amino acid residues; M° represents oxidised methionine. ⁄ in the fragment column represents the additionally identified peptides on analysis of the second injection. 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions’ intensity labelled on samples digested at 0, 15, 60 and 120 min respectively.
digested peptides are more concentrated and more easily detected and identified during the subsequent LCMS analysis. During the course of enzymatic digestion, protein is digested into large peptides and these large peptides are then further digested into small fragments. as1-Casein f15–39, which contains 25 amino acid residues was the longest iTRAQ-labelled peptide identified in the samples digested with GE at 50 °C. It was seen that the level of this peptide decreased during sampling. However, the intensity of the same sequence in the samples digested at 37 °C increased up to 60 min incubation and then decreased at 120 min presumably due to further hydrolysis of this peptide. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 0.82, 0.53 and 0.31 for the samples digested at 50 °C, respectively, and were 2.57, 3.00 and 1.28 for the samples digested at 37 °C, respectively. as1-Casein f31–55, another long iTRAQ-labelled peptide with 25 amino acid residues, was only identified in the GE digested samples at 37 °C. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 1.54, 1.48 and 0.44, respectively, which indicates that the peptide reached its highest concentration after 15 min digestion. For the GE digested samples at 50 °C, fragments of as1-casein f31–55, i.e., f31–39 and f40–55, were identified, which indicated that as1-casein f31–55 was cleaved so rapidly at peptide bond of Glu(39)-Leu(40) in the GE digested samples at 50 °C. The as1-casein f31–55 could not be detected and identified by LCMS analysis. The findings also indicate that temperature is an important factor in the digestion, e.g., incubation at 50 °C enhanced the rate of the GE digestion process. Based on the ratio of iTRAQ reporter ions (Tables 1 and 2), the concentration of 18 and 17 peptides from 27 and 28 identified
peptides reached their maximal values after 120 min digestion in the samples digested at 50 °C and 37 °C, respectively. Only two peptides (as1-casein f15–39 and as2-casein f13–23, which were specifically cleaved by GE), reached their highest concentration at 0 min in the samples digested at 50 °C. This indicates that these peptides were generated immediately after GE addition prior to sampling. The fact that 67% of the peptides identified in the samples digested at 50 °C had not reached a plateau suggests that in the iTRAQ-based quantitative analysis, protein digestion should be continued for a longer time in order to reach its maximal concentration of peptides even at the high incubation temperature, i.e., 50 °C. In the iTRAQ application protocol (Applied Biosystems), labelling of protein digests with the iTRAQ™ Reagents is recommended for 1 h at room temperature. However, peptides with partially and fully labelled iTRAQ were identified in the samples of GE digested both at 50 °C and 37 °C, i.e., as1-casein f71–89, f119–141 and as2casein f64–84 in the samples digested at 50 °C, and as1-casein f19–39 and f90–96 and as2-casein f134–145 in the samples digested at 37 °C. This result indicates that 1 h incubation at room temperature may not be a sufficient duration for completion of the iTRAQ-labelling process. Further work needs be performed to find the optimal incubation time to fully label all peptides in GE digests of a-caseins. Table 3 presents the number of phosphorylated serines and phosphorylated peptides from as1- and as2-casein identified in the iTRAQ-labelled a-caseins’ digests at 37 and 50 °C, respectively. These results of identification suggest that phosphorylated peptides were less sensitive than the non-phosphorylated peptides
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Y.-s. Zhu et al. / Food Chemistry 167 (2015) 463–467
Table 2 iTRAQ-labelled peptide sequences identified upon incubation of a-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 37 °C. Fragment
Peptide sequences
m/z (calc)
MASCOT score
115/114
116/114
117/114
f(15–30)
E-VLNENLLRFFVAPFPE-V
1025.0681 (2)
117.65
1.13
1.70
2.13
f(15–39)
E-VLNENLLRFFVAPFPEVFGKEKVNE-L
806.6994 (4)
64.8
2.57
3.00
1.28
as1-Casein
f(19–30)
E-NLLRFFVAPFPE-V
797.4491 (2)
87.02
2.29
3.75
5.38
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
692.8899 (4)
48.57
1.55
1.67
1.43
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
728.9154 (4)
57.7
1.29
1.16
1.17
f(31–39)
E-VFGKEKVNE-L
669.3870 (2)
20.63
3.56
8.30
7.67
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAME-D
841.3923 (4)
30.69
1.54
1.48
0.44
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAME-D
1121.5207 (3)
26.22
2.10
2.66
1.66
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAM°E-D
845.3911 (4)
23.08
1.54
1.41
0.80
f(40–55)
E-LSKDIGS⁄ES⁄TEDQAME-D
729.9718 (3)
31.01
0.88
0.78
1.42
f(40–55)⁄
E-LSKDIGS⁄ES⁄TEDQAM°E-D
735.3034 (3)
31.01
2.58
2.81
2.87
f(71–89)
E-IVPNS⁄VEQKHIQKEDVPSE-R
896.8038 (3)
27.69
1.88
6.69
12.73
f(71–96)
E-IVPNS⁄VEQKHIQKEDVPSERYLGYLE-Q
896.4697 (4)
21.12
1.63
2.73
2.72
f(90–96)
E-RYLGYLE-Q
529.2936 (2)
35.04
2.56
4.07
8.13
f(90–96)
E-RYLGYLE-Q
401.2322 (3)
26.67
5.59
8.56
16.56
f(111–125)
E-IVPNS⁄AEERLHSMKE-G
703.3532 (3)
47.09
2.03
2.20
1.47
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE L
773.9169 (4)
37.07
1.94
3.51
3.49
f(119–141)
E-RLHSM°KEGIHAQQKEPMIGVNQE L
777.9156 (4)
30.16
1.59
2.68
3.12
f(119–141)
E-RLHSM°KEGIHAQQKEPM°IGVNQE L
781.9143 (4)
28.33
2.13
4.08
3.83
f(126–141)
E-GIHAQQKEPMIGVNQE L
689.7014 (3)
79.82
2.51
4.15
7.62
f(126–141)
E-GIHAQQKEPM°IGVNQE L
695.0330 (3)
59.98
1.80
4.60
8.50
f(142–148)
E-LAYFYPE-L
1046.5315 (1)
31.88
4.75
7.04
11.54
f(193–199)
E-KTTMPLW
582.8381 (2)
20.01
1.48
2.13
2.97
f(13–23)
E-SIIS⁄QETYKQE-K
847.4180 (2)
60.26
1.95
5.21
3.10
f(43–51)
E-VVRNANEEE-Y
602.3079 (2)
29.87
1.78
3.88
6.08
f(64–84)
E-VATEEVKITVDDKHYQKALNE-I
752.4203 (4)
31.49
4.96
11.47
23.82
f(134–145)
E-NSKKTVDMES⁄TE-V
579.6054 (3)
20.8
1.89
4.89
5.55
f(134–145)
E-NSKKTVDMES⁄TE-V
627.6394 (3)
23.48
3.64
10.52
7.70
as2-Casein
⁄
Note: S represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labelled amino acid residues; M° represents oxidised methionine. ⁄ in the fragment column represents the additionally identified peptides in the analysis of the second injection. 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions’ intensity labelled on samples digested at 0, 15, 60 and 120 min respectively.
Table 3 Number of identified phosphorylated serine residues and phosphorylated peptides from as1- and as2-casein in the iTRAQ-labelled a-casein digests with glutamyl endopeptidase at 37 and 50 °C.
Phosphorylated serines Phosphorylated peptides
Digestion at 37 °C
Digestion at 50 °C
as1-
as2Casein
as1Casein
as2-
Casein 4 7
2 3
3 4
2 3
Casein
to LCMS detection with iTRAQ-labels. Interestingly, there was no distinct difference in digestion rates between phosphorylated and non-phosphorylated peptides (Tables 1 and 2) in GE digests of acaseins. Several peptides with reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009) were identified in the GE digested a-caseins’ samples, i.e., as1-casein f90–96, f142–148, and f193–199. The as1-casein f90–96, f142–148 were both identified in the samples digested with GE at 37 and 50 °C. The as1casein f193–199 was only identified in the samples digested with GE at 50 °C. Further research may be carried out on the bioactivity ability, e.g., ACE inhibitory, with these samples. Compared with qualitative analysis by Kalyankar et al. (2013), less sequences were identified in iTRAQ-labelled analysis. It is not surprising with the finding that each of the identified peptide includes the sequence in every sampling point. Glu(51)-Tyr(52) of as2-casein was observed to have been cleaved in the a-caseins
digested with GE at 37 and 50 °C. This result was not observed in the previous qualitative analysis. The iTRAQ-labelled as2-casein f43–50 was also identified in the samples digested at 50 °C. However, the MASCOT score was only 12 for the peptide, therefore, this sequence was not listed in Table 1. Even though the sequence was below the reliable limit for the MASCOT score, the identification of this peptide at 50 °C is supportive evidence that the Glu-Glu bond may also be hydrolysed with GE probably at a slower rate. Theoretically expected short peptides (
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Relative quantitation analysis of the substrate specificity of glutamyl endopeptidase with bovine a-caseins Yi-shen Zhu a,b,⇑, Phanindra Kalyankar b, Richard J. FitzGerald b a b
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China Department of Life Sciences, University of Limerick, Limerick, Ireland
a r t i c l e
i n f o
Article history: Received 19 February 2014 Received in revised form 6 June 2014 Accepted 6 July 2014 Available online 11 July 2014 Keywords: Glutamyl endopeptidase Bovine a-caseins’ digest Substrate specificity Isobaric tag for relative and absolute quantification LC–MS/MS
a b s t r a c t A bovine a-caseins’ preparation digested with glutamyl endopeptidase (GE) at 37 and 50 °C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Incubation temperature was shown to affect protein digestion. MS analysis of the digestion products indicated that phosphorylated peptides were less sensitive than non-phosphorylated peptides according to the MS intensities. GE hydrolysed Glu(51)-Tyr(52) and Glu(50)-Glu(51) in Glu(49)-Glu(50)-Glu(51)-Tyr(52) of bovine as1-casein. The results herein helped to confirm the precise process of a-caseins’ hydrolysis with GE, which is significant for quantifying the release of bio- and techno-functional peptides. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hydrolysed milk proteins are widely applied in food industry for many purposes, e.g., as ingredients in infant formula and in parenteral nutrition (Meisel, 2004, 2007; Nongonierma & FitzGerald, 2012; Power, Jakeman, & FitzGerald, 2013). a-Caseins (as1- and as2-casein) are major proteins in bovine milk accounting for approximately 38% of overall milk protein. Analysis of the digestion of a-caseins is therefore important for nutritional studies of dairy protein. Glutamyl endopeptidase (GE) is a chymotrypsin-like serine protease, mainly found in Bacillus species, that specifically cleaves peptide bonds between negatively charged amino acid residues (Glu/Asp) (Yokoi et al., 2001). Madsen et al. has reported that GE is more efficient in the hydrolysis of caseins compared to whey proteins (Madsen & Qvist, 1997). The substrate specificity of GE with bovine a-caseins’ preparation has been qualitatively studied (Kalyankar, Zhu, O’Keeffe, O’Cuinn, & FitzGerald, 2013). However, to our knowledge, the hydrolysis process was not quantitatively characterised. A quantitative analysis is necessary to provide more detailed information on the GE a-caseins’ hydrolysis process. Isobaric tag for relative and absolute quantification (iTRAQ) is one of the two pioneering silent isotope incorporation methodologies (Wright, Gan, Chong, & Pham, 2007). Both the identification of ⇑ Corresponding author. Tel.: +86 18900660563; fax: +86 25 58139910. E-mail address: [email protected] (Y.-s. Zhu). http://dx.doi.org/10.1016/j.foodchem.2014.07.017 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
proteins/peptides and their quantification can be achieved in a single injection (Sickmann, Burkhart, Vaudel, Zahedi, & Martens, 2011). In food science, the iTRAQ labelling was applied to quantitative proteomic analysis of bacterial enzymes in ripening cheese (Jardin, Molle, Piot, Lortal, & Gagnaire, 2012). However, quantitative analysis of enzymatic digestion of milk proteins using iTRAQ methodologies does not yet appear to have been reported in the literature. This work reports the quantitative investigation of the substrate specificity of GE with bovine a-caseins, the main proteins in bovine milk. GE was purified from Alcalase™ and its specificity was investigated using a-caseins. The hydrolysates of a-caseins digested with GE at different times at 37 and 50 °C were quantitatively analysed using iTRAQ technology. 2. Materials and methods 2.1. Materials The iTRAQÒ Reagents – 4plex Applications Kit and ICATÒ Cation Exchange Buffer Pack were supplied from Applied Biosystems (Toronto, Canada). All the other analytical reagents were obtained from Sigma Aldrich (Dublin, Ireland). 2.2. Protein purification and digestion GE was purified from Alcalase™ 2.4 L with hydrophobic interaction chromatography (HIC) of phenyl SepharoseÒ and
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ion-exchange (IEX) of HiTrap™-CM FF (Kalyankar, Zhu, O’Keeffe, O’Cuinn, & FitzGerald, 2013). The a-caseins were purified from bovine acid caseinate (Arrabawn Co-op Society Ltd, Tipperary, Ireland) following the procedure reported by Kalyankar (2011). The aqueous solution (2 mL, 1.5% (w/v)) of GE (256 nmol min 1 mL 1) digested a-caseins were incubated at 37 and 50 °C. Samples (250 lL) were withdrawn at 0, 15, 60, 120 min and were diluted with 450 lL of 0.1% (v/v) formic acid in HPLC grade H2O (Kalyankar et al., 2013). 2.3. iTRAQ labelling Each of diluted samples was labelled with iTRAQ reporter ions according to the manufacturer’s protocol (Applied Biosystems, Toronto, Canada). The brief procedure was described as: 15 lL of ethanol diluted iTRAQ reagent and 30 lL dissolution buffer were transferred to each of the 5 lL diluted samples (sampling at 0, 15, 60 and 120 min from the GE digestion); the mixture was incubated at room temperature (20 °C) for 1 h; samples were incubated for 1 h at room temperature; ethanol was removed by vacuum vortex for 1 h. The four labelled samples were mixed in a 1:1:1:1 (v/v) ratio, resulting in 27 lg of iTRAQ-labelled digest. The samples were loaded and washed on an ICATÒ cation exchange cartridge according to the manufacturer’s protocol (Applied Biosystems) to remove the excess iTRAQ reagent. 2.4. Liquid chromatography–mass spectrometry (LCMS) analysis of iTRAQ labeled GE digests of a-caseins Tandem mass spectrometry of iTRAQ-labelled samples was performed on a micrOTOFQ-II system (Bruker Daltonics, Bremen, Germany) coupled to an Ultimate 3000 nano-flow high performance liquid chromatography (HPLC) (Dionex, Sunnyvale, USA). All iTRAQ samples were first desalted online using a C18 PepMap 100 precolumn cartridge (Dionex) using 0.1% TFA at flow rate of 25 lL/min for 30 min. Desalted samples were eluted to a 15 cm, 75 mm ID C18 PepMap analytical column (Dionex) in 0.1% formic acid. Elution was then performed using a predefined 60 min LC gradient program (2–40% acetonitrile, (ACN) containing 0.1% formic acid) and further eluted for 5 min at 95% ACN. Duplicate samples of the different a-caseins’ digests were analysed on separate
LCMS/MS runs. MS measurements were all performed on a predefined 50–2400 m/z acquisition window at 2500 TOF summations. 2.5. Data analysis Tandem MS data were processed via Compass Data Analysis v 4.0 SP4 (Bruker Daltonics). Parameters of peak finder were set to S/N at least 3 and minimum 10 counts intensity. Deconvolution of MS and MS/MS charges was set to between 200 and 2500 m/z, maximum of 5+ for MS and maximum of 3+ for MS/MS spectra. All iTRAQ data were searched against the NCBInr database (downloaded at Nov 11th, 2010). The iTRAQ modifications were set to include 4-plex iTRAQ mass shifts (iTRAQ ions were labelled at Lys (K), Tyr (Y) and N-term), phosphorylation of serine and threonine (+80 Da, Ser(S)/Thr(T)) and oxidation of methionine (+16 Da, Met(M)) as variable modifications. Mass tolerances for all identifications were set to 0.06 Da for MS and 0.1 Da for MS/MS. Peptide level filters were set to a MASCOT score of 20 and at a p 6 0.05 (Zhang, Ficarro, Li, & Marto, 2009). The identified iTRAQ peptides (MS/MS) were automatically calculated using WARP-LC 1.2 (Bruker Daltonics). To detect low molecular mass peptides manually, masses were searched against theoretical peptide sequences using DataAnalysis, BioTools and Sequence Editor Software packages (Bruker Daltonics). The peptide mass and fragment mass tolerances were both set to ±0.1 Da. 3. Results In the 4-plex iTRAQ labelled peptides, the fragments released from CID were the reporter ions at 114.1, 115.1, 116.1 and 117.1 m/z. The fragmentation spectrum of f15–30 of as1-casein is shown by way of example in Fig. 1. The sequence coverage was 56% and 60% for the identified as1-casein peptides at 37 and 50 °C, respectively (Tables 1 and 2). The sequence coverage was 26% and 30% for the identified as2-casein peptides at 37 and 50 °C, respectively (Tables 1 and 2). The false discovery rates (FDR) were less than 5%. The identified sequences of the iTRAQlabelled samples were combined to provide greater coverage and precise quantification (Wright, Chong, Gan, & Pham, 2006). The sequence coverage data shows that such a high temperature during digestion may increase the extent of hydrolysis, so that the
Fig. 1. The fragment ion spectra and the sequence with identified b and y ions of iTRAQ-labelled as1-casein f15–30 in the a-caseins’ hydrolysate sample digested by glutamyl endopeptidase at 37 °C with the quantification of the iTRAQ reporter ions. Note: V indicates that the N-terminal of valine was iTRAQ-labeled.
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Y.-s. Zhu et al. / Food Chemistry 167 (2015) 463–467 Table 1 iTRAQ-labelled peptide sequences identified upon incubation of a-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 50 °C. Fragment
Peptide sequences
Ion selected for MSMS
MASCOT score
115/114
116/114
117/114
f(15–30)
E-VLNENLLRFFVAPFPE-V
1025.0681 (2)
101.23
0.81
1.13
0.60
f(15–39)
E-VLNENLLRFFVAPFPEVFGKEKVNE-L
806.6994 (4)
75.11
0.82
0.53
0.31
as1-Casein
f(19–30)
E-NLLRFFVAPFPE-V
797.4491 (2)
60.03
1.03
1.57
2.14
f(19–35)
E-NLLRFFVAPFPEVFGKE-K
766.7678 (3)
31.37
1.16
1.79
1.95
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
923.5174 (3)
119.38
1.88
1.50
0.84
f(31–39)
E-VFGKEKVNE-L
741.4380 (2)
29.13
1.57
3.18
3.33
f(40–55)⁄
E-LSKDIGS⁄ES⁄TEDQAMoE-D
735.3034 (3)
41.37
0.80
1.01
1.51
f(56–63)⁄
E-DIKQM°EAE-S
634.3257 (2)
27.18
1.74
5.08
5.75
f(71–84)⁄
E-IVPNS⁄VEQKHIQKE-D
721.0629 (3)
56.69
1.07
10.69
11.77
f(71–89)⁄
E-IVPNS⁄VEQKHIQKEDVPSE-R
848.7698 (3)
37.95
2.41
11.04
4.26
f(71–89)⁄
E-IVPNS⁄VEQKHIQKEDVPSE-R
896.8038 (3)
47.07
2.10
5.10
4.77
f(90–96)
E-RYLGYLE-Q
529.2936 (2)
34.61
1.37
4.15
5.68
f(97–110)
E-QLLRLKKYKVPQLE-I
730.1350 (3)
28.59
0.88
2.36
2.67
f(119–125)
E-RLHSM°KE-G
402.2285 (3)
21.49
1.05
2.20
2.78
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE-L
737.8914 (4)
30.1
1.08
2.42
2.92
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE-L
773.9169 (4)
61.46
1.46
2.49
3.13
f(119–141)
E-RLHSMKEGIHAQQKEPM°IGVNQE-L
777.9156 (4)
44.57
2.45
3.39
2.55
f(119–141)
RLHSM°KEGIHAQQKEPM°IGVNQE-L
781.9143 (4)
23.68
1.18
3.57
2.92
f(126–141)
E-GIHAQQKEPMIGVNQE-L
689.7014 (3)
87.91
0.80
2.67
4.34
f(142–148)
E-LAYFYPE-L
1046.5315 (1)
35.32
1.46
2.20
2.84
f(13–23)
E-S⁄IISQETYKQE-K
847.4180 (2)
55.24
0.57
0.28
0.13
f(24–33)
E-KNMAINPS⁄KE-N
548.6232 (3)
21.54
3.11
3.96
5.93 3.08
as2-Casein
f(43–51)
E-VVRNANEEE-Y
602.3079 (2)
26.34
0.86
2.31
f(64–84)⁄
E-VATEEVKITVDDKHYQKALNE-I
716.3948 (4)
22.73
1.11
2.72
1.20
f(64–84)⁄
E-VATEEVKITVDDKHYQKALNE-I
752.4203 (4)
37.38
1.00
2.33
2.45
f(69–84)
E-VKITVDDKHYQKALNE-I
620.1107 (4)
43.95
1.50
4.63
5.80
f(134–145)
E-NSKKTVDM°ES⁄TE-V
584.9370 (3)
51.06
1.03
0.89
1.66
Note: S⁄ represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labelled amino acid residues; M° represents oxidised methionine. ⁄ in the fragment column represents the additionally identified peptides on analysis of the second injection. 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions’ intensity labelled on samples digested at 0, 15, 60 and 120 min respectively.
digested peptides are more concentrated and more easily detected and identified during the subsequent LCMS analysis. During the course of enzymatic digestion, protein is digested into large peptides and these large peptides are then further digested into small fragments. as1-Casein f15–39, which contains 25 amino acid residues was the longest iTRAQ-labelled peptide identified in the samples digested with GE at 50 °C. It was seen that the level of this peptide decreased during sampling. However, the intensity of the same sequence in the samples digested at 37 °C increased up to 60 min incubation and then decreased at 120 min presumably due to further hydrolysis of this peptide. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 0.82, 0.53 and 0.31 for the samples digested at 50 °C, respectively, and were 2.57, 3.00 and 1.28 for the samples digested at 37 °C, respectively. as1-Casein f31–55, another long iTRAQ-labelled peptide with 25 amino acid residues, was only identified in the GE digested samples at 37 °C. The ratio of iTRAQ-labels, 115/114, 116/114 and 117/114, were 1.54, 1.48 and 0.44, respectively, which indicates that the peptide reached its highest concentration after 15 min digestion. For the GE digested samples at 50 °C, fragments of as1-casein f31–55, i.e., f31–39 and f40–55, were identified, which indicated that as1-casein f31–55 was cleaved so rapidly at peptide bond of Glu(39)-Leu(40) in the GE digested samples at 50 °C. The as1-casein f31–55 could not be detected and identified by LCMS analysis. The findings also indicate that temperature is an important factor in the digestion, e.g., incubation at 50 °C enhanced the rate of the GE digestion process. Based on the ratio of iTRAQ reporter ions (Tables 1 and 2), the concentration of 18 and 17 peptides from 27 and 28 identified
peptides reached their maximal values after 120 min digestion in the samples digested at 50 °C and 37 °C, respectively. Only two peptides (as1-casein f15–39 and as2-casein f13–23, which were specifically cleaved by GE), reached their highest concentration at 0 min in the samples digested at 50 °C. This indicates that these peptides were generated immediately after GE addition prior to sampling. The fact that 67% of the peptides identified in the samples digested at 50 °C had not reached a plateau suggests that in the iTRAQ-based quantitative analysis, protein digestion should be continued for a longer time in order to reach its maximal concentration of peptides even at the high incubation temperature, i.e., 50 °C. In the iTRAQ application protocol (Applied Biosystems), labelling of protein digests with the iTRAQ™ Reagents is recommended for 1 h at room temperature. However, peptides with partially and fully labelled iTRAQ were identified in the samples of GE digested both at 50 °C and 37 °C, i.e., as1-casein f71–89, f119–141 and as2casein f64–84 in the samples digested at 50 °C, and as1-casein f19–39 and f90–96 and as2-casein f134–145 in the samples digested at 37 °C. This result indicates that 1 h incubation at room temperature may not be a sufficient duration for completion of the iTRAQ-labelling process. Further work needs be performed to find the optimal incubation time to fully label all peptides in GE digests of a-caseins. Table 3 presents the number of phosphorylated serines and phosphorylated peptides from as1- and as2-casein identified in the iTRAQ-labelled a-caseins’ digests at 37 and 50 °C, respectively. These results of identification suggest that phosphorylated peptides were less sensitive than the non-phosphorylated peptides
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Y.-s. Zhu et al. / Food Chemistry 167 (2015) 463–467
Table 2 iTRAQ-labelled peptide sequences identified upon incubation of a-casein with glutamyl endopeptidase for 0, 15, 60 and 120 min at 37 °C. Fragment
Peptide sequences
m/z (calc)
MASCOT score
115/114
116/114
117/114
f(15–30)
E-VLNENLLRFFVAPFPE-V
1025.0681 (2)
117.65
1.13
1.70
2.13
f(15–39)
E-VLNENLLRFFVAPFPEVFGKEKVNE-L
806.6994 (4)
64.8
2.57
3.00
1.28
as1-Casein
f(19–30)
E-NLLRFFVAPFPE-V
797.4491 (2)
87.02
2.29
3.75
5.38
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
692.8899 (4)
48.57
1.55
1.67
1.43
f(19–39)
E-NLLRFFVAPFPEVFGKEKVNE-L
728.9154 (4)
57.7
1.29
1.16
1.17
f(31–39)
E-VFGKEKVNE-L
669.3870 (2)
20.63
3.56
8.30
7.67
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAME-D
841.3923 (4)
30.69
1.54
1.48
0.44
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAME-D
1121.5207 (3)
26.22
2.10
2.66
1.66
f(31–55)
E-VFGKEKVNELSKDIGS⁄ES⁄TEDQAM°E-D
845.3911 (4)
23.08
1.54
1.41
0.80
f(40–55)
E-LSKDIGS⁄ES⁄TEDQAME-D
729.9718 (3)
31.01
0.88
0.78
1.42
f(40–55)⁄
E-LSKDIGS⁄ES⁄TEDQAM°E-D
735.3034 (3)
31.01
2.58
2.81
2.87
f(71–89)
E-IVPNS⁄VEQKHIQKEDVPSE-R
896.8038 (3)
27.69
1.88
6.69
12.73
f(71–96)
E-IVPNS⁄VEQKHIQKEDVPSERYLGYLE-Q
896.4697 (4)
21.12
1.63
2.73
2.72
f(90–96)
E-RYLGYLE-Q
529.2936 (2)
35.04
2.56
4.07
8.13
f(90–96)
E-RYLGYLE-Q
401.2322 (3)
26.67
5.59
8.56
16.56
f(111–125)
E-IVPNS⁄AEERLHSMKE-G
703.3532 (3)
47.09
2.03
2.20
1.47
f(119–141)
E-RLHSMKEGIHAQQKEPMIGVNQE L
773.9169 (4)
37.07
1.94
3.51
3.49
f(119–141)
E-RLHSM°KEGIHAQQKEPMIGVNQE L
777.9156 (4)
30.16
1.59
2.68
3.12
f(119–141)
E-RLHSM°KEGIHAQQKEPM°IGVNQE L
781.9143 (4)
28.33
2.13
4.08
3.83
f(126–141)
E-GIHAQQKEPMIGVNQE L
689.7014 (3)
79.82
2.51
4.15
7.62
f(126–141)
E-GIHAQQKEPM°IGVNQE L
695.0330 (3)
59.98
1.80
4.60
8.50
f(142–148)
E-LAYFYPE-L
1046.5315 (1)
31.88
4.75
7.04
11.54
f(193–199)
E-KTTMPLW
582.8381 (2)
20.01
1.48
2.13
2.97
f(13–23)
E-SIIS⁄QETYKQE-K
847.4180 (2)
60.26
1.95
5.21
3.10
f(43–51)
E-VVRNANEEE-Y
602.3079 (2)
29.87
1.78
3.88
6.08
f(64–84)
E-VATEEVKITVDDKHYQKALNE-I
752.4203 (4)
31.49
4.96
11.47
23.82
f(134–145)
E-NSKKTVDMES⁄TE-V
579.6054 (3)
20.8
1.89
4.89
5.55
f(134–145)
E-NSKKTVDMES⁄TE-V
627.6394 (3)
23.48
3.64
10.52
7.70
as2-Casein
⁄
Note: S represents phosphorylated serine; the amino acid residue with dash line means it is an iTRAQ labelled amino acid residues; M° represents oxidised methionine. ⁄ in the fragment column represents the additionally identified peptides in the analysis of the second injection. 115/114, 116/114 and 117/114 represent the ratios of iTRAQ ions’ intensity labelled on samples digested at 0, 15, 60 and 120 min respectively.
Table 3 Number of identified phosphorylated serine residues and phosphorylated peptides from as1- and as2-casein in the iTRAQ-labelled a-casein digests with glutamyl endopeptidase at 37 and 50 °C.
Phosphorylated serines Phosphorylated peptides
Digestion at 37 °C
Digestion at 50 °C
as1-
as2Casein
as1Casein
as2-
Casein 4 7
2 3
3 4
2 3
Casein
to LCMS detection with iTRAQ-labels. Interestingly, there was no distinct difference in digestion rates between phosphorylated and non-phosphorylated peptides (Tables 1 and 2) in GE digests of acaseins. Several peptides with reported bioactive sequences in BIOPEP database (Dziuba & Dziuba, 2009) were identified in the GE digested a-caseins’ samples, i.e., as1-casein f90–96, f142–148, and f193–199. The as1-casein f90–96, f142–148 were both identified in the samples digested with GE at 37 and 50 °C. The as1casein f193–199 was only identified in the samples digested with GE at 50 °C. Further research may be carried out on the bioactivity ability, e.g., ACE inhibitory, with these samples. Compared with qualitative analysis by Kalyankar et al. (2013), less sequences were identified in iTRAQ-labelled analysis. It is not surprising with the finding that each of the identified peptide includes the sequence in every sampling point. Glu(51)-Tyr(52) of as2-casein was observed to have been cleaved in the a-caseins
digested with GE at 37 and 50 °C. This result was not observed in the previous qualitative analysis. The iTRAQ-labelled as2-casein f43–50 was also identified in the samples digested at 50 °C. However, the MASCOT score was only 12 for the peptide, therefore, this sequence was not listed in Table 1. Even though the sequence was below the reliable limit for the MASCOT score, the identification of this peptide at 50 °C is supportive evidence that the Glu-Glu bond may also be hydrolysed with GE probably at a slower rate. Theoretically expected short peptides (
